Characterization of Nef vesicles and their effect on T cells and macrophage

描述（由申请人提供）：感染人类免疫缺陷病毒1型（HIV-1）导致免疫系统失调和最终耗竭，称为获得性免疫缺陷综合征（AIDS）。CD4+ T细胞逐渐耗尽，受感染的个体最终死于各种机会性感染。HIV-1诱导艾滋病的确切机制尚不清楚。很明显，HIV-1感染导致CD4+ T细胞的耗竭和免疫系统的过度激活状态。然而，这些过程的确切机制仍然难以捉摸。在此应用中，我们拟探讨病毒蛋白Nef在艾滋病发病机制中可能发挥的作用。我们的初步工作表明，Nef以外泌体样囊泡的形式从HIV感染的细胞中分泌。Nef囊泡存在于感染个体的血浆中，我们的数据表明，Nef囊泡水平与抗逆转录病毒治疗后CD4+ T细胞的恢复相关。我们假设从感染细胞中以囊泡的形式分泌的Nef可以诱导对T细胞和巨噬细胞的影响，并至少导致一些在艾滋病中观察到的发病机制。在这个应用中，我们建议研究泡状Nef及其与原代T细胞和巨噬细胞的相互作用。Nef囊泡的组成将使用磁珠分离的组合来确定，然后通过流式细胞术和蛋白质组学对结合囊泡进行表征。从感染者血浆中分离的Nef囊泡将用于治疗原发性CD4 +和CD8 + T细胞和巨噬细胞。使用96孔阵列分析处理后的细胞，以确定凋亡诱导、免疫激活、细胞因子表达和基因表达。总之，收集到的信息将使我们更好地了解分泌的Nef在艾滋病发病机制中的作用。这些信息可能会导致新的治疗方法，以分泌或分泌的Nef的作用为目标。

DESCRIPTION (provided by applicant): Infection with Human Immunodeficiency Virus type 1 (HIV-1) results in dysregulation and the ultimate depletion of the immune system known as Acquired Immunodeficiency Syndrome (AIDS). CD4+ T cells are gradually depleted and the infected individuals eventually succumb to a variety of opportunistic infections. The exact mechanisms of how HIV-1 induces AIDS are not well understood. It is clear that infection with HIV-1 results in depletion of CD4+ T cells and a state of hyperactivation of the immune system. However, the exact mechanism of these processes remains elusive. In this application, we propose to explore what role the viral protein Nef might play in AIDS pathogenesis. Our preliminary work shows that Nef is secreted from HIV infected cells in the form of exosome-like vesicles. Nef vesicles are present in the plasma of infected individuals and our data suggests Nef vesicle levels correlate with recovery of CD4+ T cells after antiretroviral therapy. We hypothesize that Nef, secreted from infected cells in the form of vesicles can induce effects on T cells and macrophage and cause at least some of the pathogenesis observed in AIDS. In this application we propose to study vesicular Nef and its interaction with primary T cells and macrophage. The composition of Nef vesicles will be determined using a combination of magnetic bead separation followed by characterization of bound vesicles by flow cytometry and Proteomics. Nef vesicles isolated from the plasma of infected individuals will be used to treat primary CD4 + and CD8 + T cells and macrophage. The treated cells will be analyzed to determine induction of apoptosis, immune activation, cytokine expression and gene expression using 96-well arrays. Together the information gathered will give us a better picture of the role of secreted Nef in the pathogenesis of AIDS. Such information could lead to new therapeutic approaches that target secretion or the action of secreted Nef. PUBLIC HEALTH RELEVANCE: HIV-AIDS is disease characterized by the destruction of normal immunity. In this application we propose that infection with HIV causes the release of large numbers of virus-like packets called "exosomes". Our theory is that release of exosomes is responsible for at least some of the loss of immune function seen in AIDS.